Laminin 521 Stabilizes the Pluripotency Expression Pattern of Human Embryonic Stem Cells Initially Derived on Feeder Cells

Abstract

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.

Figure 1

hES cells derived on hFFs and cultured to p4 on LN521. All cell lines (HS360, HS364, HS380, HS401, and HS420) cultured on LN521 exhibited typical hES cell morphology at p4 (a). The hierarchical clusters of gene expression levels for all the cell lines analysed by TLD arrays show a heterogeneous expression profile specific for every cell line. Red colour represents high gene expression (low dCT value) and green colour represents low gene expression (high dCT value) (b). As shown by the Venn diagram, all five cell lines exhibited higher expression of nine stemness-related genes and lower expression of four genes related to differentiation (c).

Figure 2

hES cells derived on hFFs and cultured to p9 on LN521. All five cell lines transferred from hFFs onto LN521 exhibited normal hES cell morphology (a). Immunofluorescence analysis of NANOG (b), POU5F1 (c), SOX2 (d), SSEA4 (e), and TRA-1-60 (f) expression, shown for HS380 as a representative cell line, revealed the presence of pluripotency markers after p9 on LN521 (red staining: NANOG, POU5F1, and SOX2; green staining: SSEA4 and TRA-1-60; and blue staining: DAPI). Scale bars: 50 μm. Immunofluorescence analysis of the expression of differentiation markers for endoderm (AFP) (g), ectoderm (TUJ1) (h), and mesoderm (alpha SMA) (i), shown in HS380 as a representative cell line, demonstrates the potential of hES cells cultured on LN521 to differentiate spontaneously into the three germ layers (green staining: AFP, alpha SMA and TUJ1; blue staining: DAPI). Scale bars: 50 μm.

Figure 3

Homogenization of the expression of pluripotency genes and genes related to male germ cells and somatic gonadal cells after prolonged culture of hES cells on LN521 presented as heat maps for dCT values on hFFs at p4 and LN521 at p4 and p9. Red colour represents high gene expression (low dCT value) and green colour represents low gene expression (high dCT value); undetected (low/no expression) samples was given a dCT value of 15. (Below) a table of the mean and SD values of the five cell lines showing the relative expression values of pluripotency genes and genes related to stemness and male gonadal cells to GAPDH. The coefficient of variation is calculated as SD/mean and the variations among the lines are classified into four groups: less than 10% (green), 11% to 25% (yellow), 26%–50% (orange), and more than 50% (red).